Microscopy, Culturing, and Counting (Lab)

Question 1

What is resolution?

Answer 1

The ability to distinguish separate objects as actually being separate

Question 2

How do you improve resolution?

Answer 2

Use a smaller wavelength

Question 3

Are smaller or larger resolutions better?

Answer 3

Smaller

Question 4

Why do we use oil immersion?

Answer 4

Causes the light to travel in a straight path instead of being refracted by the air, which causes more light to enter the objective

Question 5

Why do we use Koehler illumination?

Answer 5

Provides the best possible image by setting up the light path correctly

Question 6

What are the 2 purposes of aseptic technique?

Answer 6

To protect yourself and the environment from your experiment
To protect your experiment from yourself and the environment

Question 7

What are 5 ways to do aseptic technique?

Answer 7

1. Wash your hands
2. Ethanol the bench
3. Keep lids on
4. Don’t put lids down on contaminated surfaces
5. Flame culture tubes

Question 8

Why do we streak plates for single colonies?

Answer 8

Allows us to purify colonies to get a culture of genetically identical cells

Question 9

What are 2 ways to sterilize stuff?

Answer 9

Dry heat: flaming with bunsen burners

Moist heat: autoclave - high pressure and heat

Question 10

Which method of sterilization can kill endospores? Dry heat or moist heat?

Answer 10

Moist heat (the autoclave)

Question 11

Are plates labelled on the lid or the bottom?

Answer 11

Bottom. Labelling on the lid would become difficult if the lids accidentally became switched

Question 12

Why do we do serial dilutions?

Answer 12

Determine the appropriate dilution that gives between 30-300 colonies when plated, then using that to calculate the original cell density

Question 13

Why would we use a pure liquid culture over a pure culture on an agar plate?

Answer 13

Much easier to get DNA or proteins out of a cell suspension than a plate

Question 14

What are the 6 different types of medium?

Answer 14

1. Defined
2. Minimal
3. Complex
4. Enriched
5. Selective
6. Differential

Question 15

What is defined medium?

Answer 15

We know the exact nutrient composition of both organic and inorganic compounds

Question 16

What is minimal medium?

Answer 16

A specific type of defined medium where only prototrophic cells can grow. Contains inorganic salts, ions, and a carbon source. Everything else needs to be synthesized de novo by the cells

Question 17

What is complex medium?

Answer 17

Highly nutritious medium full of organic and inorganic energy sources made from a digest of plant, animal, or microbial products (Casein, soy, yeast, beef). Don’t know the exact nutrient composition

Question 18

What is enriched medium?

Answer 18

Complex medium + a few other things like blood or serum that supports the growth of fastidious microorganisms

Question 19

What is selective medium?

Answer 19

Medium that only allows the growth of certain microorganisms

Question 20

What is differential medium?

Answer 20

Complex medium containing some sort of dye that will allow different properties to be distinguished

Question 21

Why is all growth medium somewhat selective?

Answer 21

No medium will support the growth of every single bacterial species

Question 22

What are endospores?

Answer 22

A differentiated dormant cell type produced by some species that are resistant to desiccation, radiation, heat

Question 23

How do you break the dormancy on an endospore?

Answer 23

High heat treatment

Question 24

What are total cell counts?

Answer 24

All cells are counted, whether alive, dead, or dormant

Question 25

What are viable cell counts?

Answer 25

Only cells that are alive and capable of forming colonies are counted

Question 26

What are 3 direct ways to get a total cell count?

Answer 26

1. Microscopy to look at the cells
2. Hemocytometer without methylene blue
3. Petroff-Hausser counter

Question 27

What are 3 indirect ways to get a total cell count?

Answer 27

1. Turbidity measurements with OD
2. Dry weight
3. Measurements of cell components (ex. DNA)

Question 28

What is the direct way to get a viable cell count?

Answer 28

Hemocytometer with methylene blue

Question 29

What are 2 indirect ways to get a viable cell count?

Answer 29

1. Serial dilution and plate count

2. Measurement of incorporation of an indicator molecule

Question 30

How do you calculate the original viable cell concentration from a plate count? What are the units?

Answer 30

Average # of colonies on a plate/dilution factor x volume of dilution plated
Units are CFU/ml

Question 31

Why can plate counts sometimes be inaccurate?

Answer 31

One colony didn’t necessarily stem from 1 cell. Lots of bacteria show clumping, and it is impossible to distinguish different colonies

Question 32

Why does the methylene blue dye stain alive and dead cells differently?

Answer 32

Dead cells have compromised membranes, so the dye can get in

Question 33

How do you get a viable cell count from a hemocytometer?

Answer 33

of alive cells - # of dead cells/# of total cells